CN114774296B - Pichia pastoris strain HGC34 resistant to high Wen Jiye and application thereof in deodorization and degradation of livestock manure - Google Patents
Pichia pastoris strain HGC34 resistant to high Wen Jiye and application thereof in deodorization and degradation of livestock manure Download PDFInfo
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- 238000006731 degradation reaction Methods 0.000 title abstract description 10
- 241000235048 Meyerozyma guilliermondii Species 0.000 claims abstract description 28
- 239000002361 compost Substances 0.000 claims abstract description 22
- 230000000813 microbial effect Effects 0.000 claims description 50
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F11/00—Treatment of sludge; Devices therefor
- C02F11/02—Biological treatment
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- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
- C05F17/20—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation using specific microorganisms or substances, e.g. enzymes, for activating or stimulating the treatment
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/20—Nature of the water, waste water, sewage or sludge to be treated from animal husbandry
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2303/00—Specific treatment goals
- C02F2303/02—Odour removal or prevention of malodour
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Abstract
The invention belongs to the technical field of microorganisms, and provides a high Wen Jiye resistant pichia pastoris strain HGC34 and application thereof in deodorization and degradation of livestock and poultry manure. The pichia guilliermondii (meyerozymekuilibermondii) HGC34 was preserved in the chinese collection of typical cultures at 10 and 31 of 2016 with a preservation number of cctcno: m2016600. The Gao Wenji-resistant pichia pastoris HGC34 has high temperature resistance, can remarkably remove odor in livestock and poultry manure such as chicken manure, pig manure and the like, and achieves the purposes of protecting environment and promoting rapid degradation and decomposition of compost.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a high Wen Jiye resistant pichia pastoris strain HGC34 and application thereof in deodorization and degradation of livestock and poultry manure.
Background
With the large-scale and facility rapid development of domestic breeding industry, livestock and poultry breeding generates a large amount of wastes, and serious environmental and social problems are brought. Odor is one of the challenges faced by the centralized treatment of livestock and poultry manure. The main components of the odor generated in the composting process comprise ammonia, hydrogen sulfide and Volatile Organic Compounds (VOCs), which cause serious harm to the ecological environment. The various microbial communities in the compost habitat play an important role in odor generation and deodorization, some microbial communities can generate odor by decomposing organic matters, some microorganisms can utilize odor components to finish self metabolism, and in addition, the microbial communities are continuously replaced when the heap temperature changes.
NH in chicken manure and pig manure compost is removed by utilizing high-efficiency deodorizing bacterial strain and cellulose decomposing bacterial colony 3 And H 2 S has related researches and reports, and the in-situ removal of odor in livestock manure compost by a microbiological method is proved to be efficient and feasible. The microorganism with deodorizing and degrading functions in the nature is various, and the development of more efficient deodorizing and degrading strains is a technical problem to be solved urgently, and the high-quality deodorizing and degrading strains also provide high-quality strain resources for removing odor substances in the livestock and poultry manure by a biological method.
Disclosure of Invention
The invention aims to provide a high Wen Jiye resistant pichia pastoris strain HGC34 and application thereof in livestock manure deodorization degradation, and the Gao Wenji resistant pichia pastoris strain HGC34 can remarkably remove odor in livestock manure such as chicken manure, pig manure and the like, so as to achieve the purposes of protecting environment and promoting rapid degradation and decomposition of compost.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides pichia guilliermondii (Meyerozyma guilliermondii) HGC34, wherein the pichia guilliermondii HGC34 is preserved in China Center for Type Culture Collection (CCTCC) in the 10 th month 31 th 2016, and the preservation number is CCTCC NO: m2016600.
The invention provides a microbial deodorant, and the active ingredients of the microbial deodorant comprise pichia pastoris HGC34.
Further, the microbial deodorant has a bacterial concentration of 1.0X10 of Pichia guilliermondii HGC34 9 ～8.0×10 9 CFU/mL。
The invention also provides a method for reducing the odor of the livestock manure compost by using the microbial deodorant, wherein the method for reducing the odor is to add the microbial deodorant into the livestock manure compost for treatment; the ratio of the microbial deodorant to the livestock manure is that the microbial deodorant to the livestock manure=2-8 mL and 80-120 kg.
Further, the water content of the livestock manure is 40-50%.
Further, the water content of the livestock manure is regulated by adding waste fungus chaff with the water content of less than 30% into the livestock manure; the addition amount of the waste fungus chaff is 1-5% of the mass of the livestock manure.
Further, the temperature of the treatment is 25-45 ℃, the pH value of the treatment is 5.5-6.5, and the temperature of the livestock manure composting center is 40-65 ℃.
Compared with the prior art, the invention has the beneficial effects that:
the Gao Wenji resistant pichia pastoris HGC34 provided by the invention is separated from natural environment, and has no biological danger; and the Gao Wenji-resistant pichia pastoris HGC34 is recorded in the embodiment, and the live cell percentages of the strains in the liquid are 93.1%,88.6% and 82.4% respectively under three conditions of 55 ℃,60 ℃ and 65 ℃, which indicates that the pichia pastoris HGC34 can resist the high temperature of 65 ℃ for survival.
According to the invention, the Gao Wenji resistant pichia pastoris HGC34 is added into the livestock manure, so that the odor in the livestock manure such as chicken manure, pig manure and the like can be obviously removed, the purposes of protecting the environment and promoting the rapid degradation and decomposition of the compost are achieved.
Drawings
FIG. 1 is a sample of rotten chestnut isolated from Pichia pastoris HGC 34;
FIG. 2 is a diagram of agarose gel electrophoresis detection of PCR products, M: dnamarker dl5000;
FIG. 3 is a plot of Pichia pastoris HGC34 after 20 days post treatment at 5% inoculum size;
FIG. 4 shows growth of Pichia pastoris HGC34 at different temperature conditions;
FIG. 5 shows the morphology of Pichia pastoris HGC34 cells.
Preservation description
Pichia guilliermondii HGC34, latin Meyerozyma guilliermondii, deposited at China center for type culture collection at the address: china, university of Wuhan, and the preservation date is 2016, 10 and 31 days, and the preservation number is CCTCC NO: m2016600.
Detailed Description
The invention provides pichia guilliermondii (Meyerozyma guilliermondii) HGC34, wherein the pichia guilliermondii HGC34 is preserved in China Center for Type Culture Collection (CCTCC) in the 10 th month 31 th 2016, and the preservation number is CCTCC NO: m2016600.
In the invention, the sequence of the ITS region of the 18S rDNA gene of the Pichia guilliermondii HGC34 is shown as SEQ NO. 1; the pichia guilliermondii HGC34 is isolated from extremely rotten chestnut; pichia guilliermondii HGC34 has round or oval cell shape under microscope, is transparent, slightly bigger than bacteria, has no flagellum and hypha, and has white living cells, blue dead cells, visible bud, and a smaller bud beside a larger parent cell.
The invention provides a microbial deodorant, and the active ingredients of the microbial deodorant comprise pichia pastoris HGC34.
In the invention, the microbial deodorant has the bacteria concentration of Pichia guilliermondii HGC34 of 1.0x10 9 ～8.0×10 9 CFU/mL, preferably 2.0X10 9 ～6.0×10 9 CFU/mL, more preferably 3.0X10 9 ～5.0×10 9 CFU/mL。
The preparation method of the deodorant bacterial agent comprises the steps of carrying out activation culture on pichia pastoris HGC34 in YPA liquid culture medium for 3-5 days, and counting by microscopic observation to obtain the product with the viable count of 1.0X10 9 ～8.0×10 9 CFU/mL。
The invention also provides a method for reducing the odor of the livestock manure compost by using the microbial deodorant, wherein the method for reducing the odor is to add the microbial deodorant into the livestock manure compost for treatment.
In the invention, the ratio of the microbial deodorant to the livestock manure is microbial deodorant to livestock manure=2-8 mL to 80-120 kg, preferably microbial deodorant to livestock manure=3-7 mL to 85-115 kg, and more preferably microbial deodorant to livestock manure=4-6 mL to 90-110 kg; the microbial deodorant is sprinkled into the livestock manure according to the adding proportion, and the microbial deodorant and the livestock manure are uniformly mixed by adopting a conventional uniform mixing method.
In the invention, the water content of the livestock manure is 40-50%, preferably 42-48%, and more preferably 44-46%.
In the invention, the water content of the livestock manure is regulated by adding the waste fungus chaff into the livestock manure; the water content of the waste fungus chaff is below 30%, preferably 25-28%, more preferably 26-27%; the addition amount of the waste fungus chaff is 1-5% of the mass of the livestock manure, preferably 2-4%, and more preferably 2.5-3.5%.
In the method for reducing odor, a microbial deodorant is added into livestock manure compost for treatment, and the treatment temperature is 25-45 ℃, preferably 30-40 ℃, and more preferably 32-38 ℃; the pH of the treatment is 5.5 to 6.5, preferably 5.7 to 6.3, more preferably 5.9 to 6.1; the temperature of the livestock manure composting center is 40-65 ℃, preferably 45-62 ℃, and more preferably 50-60 ℃.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Experimental example 1
Collecting the strain in the extremely rotten chestnut sample (figure 1), separating and identifying, extracting genome DNA from the separated strain according to a conventional method, and performing PCR amplification after obtaining the genome DNA, wherein the primer sequence of the ITS region sequence of the 18S rDNA gene is as follows: ITS1 (SEQ: no. 2): 5'-TCCGTAGGTGAACCTGCGG-3'; ITS4 (SEQ: no. 3): 5'-TCCTCCGCTTATTGATATGC-3'. After amplification, the PCR products were detected by agarose gel electrophoresis, and the electrophoresis pattern is shown in FIG. 2. The sequenced genomic DNA was placed into blast for comparison, and the results showed that HGC34 and Meyerozyma guilliermondii (Pichia guilliermondii) had the closest affinity. The strain obtained by identification and separation is pichia guilliermondii.
Experimental example 2
The separated Pichia pastoris HGC34 is put into a test tube with fresh chicken manure as a bottom material, a laboratory test is performed on the strain application, whether the odor substances exist or not and the degree are judged through smell every 24 hours, and the experimental design is repeated. Odor intensity ratings are as in table 1:
table 1 odor evaluation rating
Deodorizing seed solution of cultured HGC34 strain (strain concentration of 8.0X10) at inoculum concentration of 0.5%, 1.0%, 3.0% and 5.0%, respectively 9 CFU/mL) were inoculated into fresh chicken manure-containing test flasks, stirred well, incubated in a constant temperature incubator at 30 ℃ and used as a blank control group (CK), and the effect of the inoculum size on deodorization performance was observed every 10 days, and the results are shown in table 2.
TABLE 2 Effect of different inoculum sizes on deodorization
As can be seen from table 2, the seed solution deodorized by pichia pastoris HGC34 in the inoculation season is completely deodorized according to the inoculation amount of 5.0% for 20d, and the state of the seed solution deodorized by pichia pastoris HGC34 in the inoculation amount of 5.0% after 20 days of treatment is shown in fig. 3; the full deodorization time of 3% of the inoculation amount is prolonged to 30 days, and the blank control group 50d still has strong odor, which shows that the deodorization microbial inoculum has obvious deodorization effect on the tested fresh chicken manure.
Experimental example 3
In YPD liquid culture medium test tubeIn the above step, seed solution of HGC34 strain was inoculated at an inoculum size of 3.0% (bacterial solution concentration 6.8X10) 8 CFU/mL), at 30 ℃,35 ℃,40 ℃,45 ℃,50 ℃,55 ℃,60 ℃,65 ℃ for 48 hours, and calculating the number of saccharomycetes and the condition of dead living cells by a blood cell counting plate. As shown in FIG. 4, HGC34 grew best at 55℃indicating that the strain was a thermophilic bacterium. The pichia pastoris HGC34 is observed by a merocyanin staining method, and the cell shape is round or oval under a microscope, is transparent and slightly larger than bacteria. No flagella, no hyphae, mostly white, a few blue, visible budding, a smaller bud beside the larger parent cell (see fig. 5). Counting the condition of dead living cells under a microscope, wherein the dead cells are blue, the living cells are colorless, and the proportion of the dead living cells can be calculated by counting. The results show that the percentages of the live cells of the strain in the liquid are 93.1%,88.6% and 82.4% respectively under the conditions of 55 ℃,60 ℃ and 65 ℃, which indicates that the pichia guilliermondii HGC34 can resist the high temperature of 65 ℃ for survival.
Experimental example 4
The production and application test of the achievements is carried out in a biological organic fertilizer fermentation workshop of the Hubei championship limited company. Adopting pile type composting fermentation, taking fresh chicken manure as main raw material, adding waste Lentinus edodes residue according to 5% to adjust composting state, adjusting water content to 55%, adding HGC34 bacterial agent according to 3% (bacterial liquid concentration is 8.0X10) 9 CFU/mL), 1kg of the seal was sampled every 10d from 20cm below the compost surface layer, and the deodorizing condition was evaluated by sniffing in a well ventilated laboratory with the evaluation scale of table 1. The results showed that the odor grade of the compost sample to which the microbial inoculum was added was 3 grade, the odor intensity was 1 grade after 20d, and the odor intensity was 0 grade after 30 d. And the odor intensity of the control sample without the microbial inoculum is still 4 grade after 30d, which shows that the deodorizing effect of the HGC34 microbial inoculum is obvious.
Example 1
The embodiment provides a method for reducing livestock manure compost odor by using a microbial deodorant, which comprises the following steps:
(1) Pretreatment of livestock manure: collecting fresh chicken manure with the water content of 70%, and adding 3% of waste fungus chaff with the water content of 25% into the chicken manure to adjust the water content of the chicken manure to 45%;
(2) Preparation of microbial deodorant: the microbial deodorant comprises Pichia guilliermondii HGC34, wherein the concentration of the Pichia guilliermondii HGC34 is 1.0X10% 9 CFU/mL；
(3) Uniformly mixing the microbial deodorant and the livestock manure according to the ratio of microbial deodorant to livestock manure=5 mL to 100kg, and carrying out natural composting, wherein the ambient temperature of the composting is 35 ℃, the composting pH is 5.5, the composting center temperature is 50 ℃, the composting is carried out once in 3 days, and the composting is carried out for 3 times continuously. The control group was not added with microbial agent.
Example 2
The embodiment provides a method for reducing livestock manure compost odor by using a microbial deodorant, which comprises the following steps:
(1) Pretreatment of livestock manure: collecting fresh pig manure with the water content of 65%, and adding waste fungus chaff with the water content of 20% and 2% into the pig manure to adjust the water content of the pig manure to 50%;
(2) Preparation of microbial deodorant: the microbial deodorant comprises pichia guilliermondii HGC34, wherein the concentration of the pichia guilliermondii HGC34 is 3.0x10 9 CFU/mL；
(3) Uniformly mixing the microbial deodorant and the livestock manure according to the ratio of microbial deodorant to livestock manure=6 mL to 110kg, and carrying out natural composting, wherein the ambient temperature of the composting is 30 ℃, the composting pH is 6.0, the composting center temperature is 55 ℃, the composting is carried out once in 3 days, and the composting is carried out for 3 times continuously. The control group was not added with microbial agent.
Example 3
The embodiment provides a method for reducing livestock manure compost odor by using a microbial deodorant, which comprises the following steps:
(1) Pretreatment of livestock manure: collecting fresh chicken manure with the water content of 75%, and adding 5% of waste fungus chaff with the water content of 27% into the chicken manure to adjust the water content of the chicken manure to 50%;
(2) Preparation of microbial deodorant: by a means ofThe microbial deodorant comprises Pichia guilliermondii HGC34, wherein the concentration of the Pichia guilliermondii HGC34 is 2.5X10 9 CFU/mL；
(3) Uniformly mixing the microbial deodorant and the livestock manure according to the ratio of microbial deodorant to livestock manure=4 mL to 90kg, and naturally composting at the ambient temperature of 40 ℃, the composting pH of 6.5 and the composting center temperature of 60 ℃, turning the compost once for 3 days, and continuously turning the compost for 3 times. The control group was not added with microbial agent.
The application effects of the microbial deodorant bacterial agent in the above 3 examples on the treatment of livestock and poultry manure are shown in table 3.
TABLE 3 application effects of microbial deodorant to livestock and poultry feces treatment
From the above examples and experimental examples, the invention provides the high Wen Jiye resistant pichia pastoris strain HGC34 and the application thereof in deodorizing and degrading livestock manure, and the Gao Wenji resistant pichia pastoris HGC34 can remarkably remove odor in livestock manure such as chicken manure, pig manure and the like, thereby achieving the purposes of protecting environment and promoting rapid degradation and decomposition of compost.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
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Claims (7)
1. Pichia guilliermondii (Meyerozyma guilliermondii) HGC34, wherein said Pichia guilliermondii HGC34 was deposited at China center for type culture collection (CCTCC NO) at 10 and 31 of 2016: m2016600.
2. A microbial deodorant bacterial agent, wherein the active ingredient of the microbial deodorant bacterial agent comprises pichia pastoris HGC34 according to claim 1.
3. The microbial deodorant according to claim 2, wherein the microbial deodorant comprises a bacterial concentration of 1.0×10 pichia guilliermondii HGC34 9 ～8.0×10 9 CFU/mL。
4. A method for reducing the odor of livestock manure compost by using the microbial deodorant according to claim 2, which is characterized in that the method for reducing the odor of livestock manure compost is to add the microbial deodorant into the livestock manure compost for treatment; the ratio of the microbial deodorant to the livestock manure is that the microbial deodorant to the livestock manure=2-8 mL and 80-120 kg.
5. The method according to claim 4, wherein the water content of the livestock manure is 40-50%.
6. The method according to claim 5, wherein the water content of the livestock manure is adjusted by adding waste fungus chaff with the water content of less than 30% to the livestock manure; the addition amount of the waste fungus chaff is 1-5% of the mass of the livestock manure.
7. The method according to claim 4, wherein the temperature of the treatment is 25-45 ℃, the pH value of the treatment is 5.5-6.5, and the temperature of the livestock manure composting center is 40-65 ℃.
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| CN113501893A (en) * | 2021-06-21 | 2021-10-15 | 中国热带农业科学院橡胶研究所 | Environment-friendly natural rubber processing method |
| CN114015586A (en) * | 2021-12-01 | 2022-02-08 | 中国科学院成都生物研究所 | Deodorizing and degrading composite microbial inoculum as well as preparation method and application thereof |
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| CN113501893A (en) * | 2021-06-21 | 2021-10-15 | 中国热带农业科学院橡胶研究所 | Environment-friendly natural rubber processing method |
| CN114015586A (en) * | 2021-12-01 | 2022-02-08 | 中国科学院成都生物研究所 | Deodorizing and degrading composite microbial inoculum as well as preparation method and application thereof |
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